S185: TARGETING DNA2 OVERCOMES METABOLIC REPROGRAMMING IN 1Q21 MULTIPLE MYELOMA
Authors: Natthakan Thongon, Feiyang MA, Pamela Lockyer, Natalia Baran, Jintan Liu, Caleb Class, Lin Tan, Paola Storti, Valentina Marchica, Irene Ganan-Gomez, Bethany Wildeman, Vera Adema, Philip Lorenzi, Rodrigo Fonseca, Marta Chesi, Elisabeth Manasanch, Andrea Viale, Simona Colla
Journal: HemaSphere (2023)

Topic: 13. Myeloma and other monoclonal gammopathies - Biology & Translational Research Background: DNA damage resistance is a major barrier to effective DNA-damaging anticancer therapy in multiple myeloma (MM). Our previous studies of recurrently amplified 1q21 genes in MM have identified ILF2 (interleukin enhancer binding factor 2) as a key modulator of the DNA repair pathway, which promotes adaptive mechanisms to genotoxic stress in a dose-dependent manner, explaining why 1q21 patients benefit less from high-dose therapy than non-1q21 patients (Cancer Cell, 2017). Aims: To discover novel mechanisms through which MM cells overcome DNA damage, we investigated how MM cells became resistant to antisense oligonucleotide therapy targeting ILF2. Methods: We used antisense oligonucleotides targeting ILF2 (ILF2 ASOs) as a tool to deplete ILF2 expression and induce DNA damage activation in 5 MM cell lines. We performed functional validation studies using the most effective ILF2 ASOs among the 300 screened and confirmed that depleting ILF2 induced DNA activation in MM cell lines and reduced tumor burden in MM xenografts. However, MM cells eventually became resistant to ILF2 depletion and overcame DNA damage activation after long exposure to ILF2 ASOs for 3 weeks. We used a CRISPR/Cas9 screening strategy targeting 196 DNA repair genes to identify DNA repair proteins that, when depleted, overcame resistance to ILF2 ASOs. To dissect the molecular mechanisms of the DNA damage resistance, we performed RNA-seq, scRNA-seq, metabolomic, seahorse, and transmission electron microscopy (TEM) analyses. Results: scRNA-seq revealed that ILF2 ASO resistant cells had significantly upregulated genes involved in oxidative phosphorylation (OXPHOS), DNA repair, and reactive oxidative species (ROS) pathways. The upregulation of the OXPHOS pathway was confirmed by metabolomic analysis. Consistent with these results, ILF2 ASO-resistant MM cells were significantly more sensitive to the OXPHOS inhibitor IACS-010759 than ILF2 ASO-sensitive cells. Using the CRISPR/Cas9 screening strategy targeting DNA repair genes, we identified the DNA repair protein DNA2, whose loss of function suppressed MM cells’ ability to overcome ILF2 ASO-induced DNA damage. We demonstrated that the DNA2 inhibitor NSC105808 (NSC) significantly enhanced ILF2-ASO-induced apoptosis in ILF2 ASO-resistant MM cells and xenografts. Inhibition of DNA2 activity significantly decreased the oxygen consumption rate while increasing ROS production in only ILF2-depleted cells. TEM analysis showed that NSC-treated ILF2-depleted cells had fragmented mitochondrial cristae structures, whose perturbations can affect the OXPHOS system and impair cell metabolism. A shift from glycolysis to high mitochondrial energy metabolism is sufficient to promote proteasome inhibitor (PI) resistance in MM. Inhibition of DNA2 significantly decreased the survival of plasma cells isolated from bone marrow of MM patients who relapsed after PI treatment. These data suggest that DNA2 is essential to counteracting oxidative DNA damage and maintaining mitochondrial respiration in the cells which have high mitochondria demand. Summary/Conclusion: Our study revealed a unique vulnerability of MM cells that are forced to use OXPHOS to overcome DNA damage activation. Given that metabolic reprogramming is a hallmark of cancer progression, further studies will clarify whether therapeutically targeting DNA2 has a broad spectrum of anti-cancer applications.Keywords: Multiple myeloma, DNA repair, Resistance, DNA damage